Method for treating cancer

ABSTRACT

The present invention relates to a method for the treatment of cancer in a subject which comprises the steps of: (i) administering electroporation to a tumour in the subject; and (II) administering a T or B-cell activiating agent to the subject, wherein step (i) and (II) may be performed in either order.

FIELD OF THE INVENTION

The present invention relates to a method for treating cancer in asubject.

BACKGROUND TO THE INVENTION

Electrochemotherapy (ECT) is a local treatment of cancer, which combinesthe use of a medical device with pharmaceutical agents to achieve localtumour control in solid cancers. The procedure consists of applyingshort high-intensity pulsed electric fields to cells, in response towhich the plasma membrane's permeability to various moleculestransiently increases. This facilitates cellular uptake of cytotoxicagents, thus increasing their cytotoxicity. The treatment is based onelectroporation, which occurs when an externally delivered electricfield induces a sufficiently large transmembrane voltage.Electroporation is, in addition to its use in electrochemotherapy, usedalso as non-viral gene delivery method to cells in vitro and invivo—gene electrotransfer. Furthermore, electroporation as sole modalitycan be used as tumour ablation in the form of irreversibleelectroporation, also referred to as non-thermal irreversibleelectroporation.

Applications for electrochemotherapeutic-based treatment of cutaneousand subcutaneous tumours using drugs such as bleomycin and cisplatinhave reached clinical use. ECT with bleomycin was used to treat apatient for the first time in 1991, while ECT with cisplatin was usedfor the first time in 1995. Multiple positioning of the electrodes, andsubsequent pulse delivery, can be performed during a session to treatthe whole lesion, provided that drug concentration is sufficient.Treatment can be repeated over the course of weeks or months to achieveregression of large lesions.

In a number of clinical studies (phase II and phase III), investigatorshave concluded that ECT of cutaneous or subcutaneous metastasis ortumours with bleomycin and cisplatin have an objective response rate ofmore than 80%. Reduction of tumour size has been achieved withelectrochemotherapy faster and more efficiently than in standardchemotherapy for both cutaneous and subcutaneous tumours.

Whilst studies have shown that ECT is effective at achieving localtumour reduction and potential tumour resolution, it is largelyincapable of generating a systemic antigen specific T/B cell immuneresponse. This incapability can lead to disease reoccurrence.

There is thus a need for a cancer therapy, which is not associated withthese issues.

DESCRIPTION OF THE FIGURES

FIG. 1: Growth curve of ECT combined with ICOS in CT26 model

Representative CT26 tumor growth curve. Each Balb/C mouse wassubcutaneously injected with 1×10⁶ CT26 cells in the flank. When tumoursreached an approximate size of 100 mm³ they were treated withelectroporation only (▪), electrochemotherapy only (▴),electrochemotherapy combined with ICOS (●) or untreated (●). 6mice/groups were used and the experiment was performed twice. Tumourvolume was calculated using the formula V=ab²π/6. Data is presented asthe means±standard error of the mean.

FIG. 2: Kaplan-Meier survival curve ECT combined with ICOS in CT26 model

Representative Kaplan-Meier survival curve of CT26 treated tumours wasmeasured. Only mice treated with ICOS/ECT combination survived. 40% ofmice survived and were still alive at approx. 200 days. All other groupswere sacrificed by day 38.

FIG. 3: Growth curve of ECT combined with ICOS in B16F10 model

Representative growth curve of B16F10 tumour. Each C57BL/6J wassubcutaneously injected with 2×10⁵ B16F10 cells in the flank of themice. When the tumours grew to an approximate size of 100 mm³ they weretreated with electroporation only (▪), electrochemotherapy only (▴),electrochemotherapy combined with ICOS (♦) ICOS only (▾) or untreated(●). 6 mice/groups were used and the experiment was performed twice.Tumor volume was calculated using the formula V=ab²π/6. Data ispresented as the means±standard error of the mean.

FIG. 4: Kaplan-Meier survival curve ECT combined with ICOS in B16F10model

Representative Kaplan-Meier survival curve of B16F10 treated tumours wasmeasured. Mice treated with ICOS/ECT combination survived to 48 days.All other groups were sacrificed by day 25.

FIG. 5: Lung metastases burden

Measured lung weight in lung metastatic cancer model.

FIG. 6: Programmed Cell Death Receptor 1 (PD-1) and Programmed CellDeath Ligand (PD-L1) expression

PD-1 and PD-L1 expression within tumour tissue.

FIG. 7: Flow cytometry (1)

Flow cytometry analysis of excised tumour tissue.

FIG. 8: Flow cytometry (2)

FIG. 8 presents a repeat of the flow cytometry data limited to therelevant iCOS groups: CD8+ T cells, B220, CD138.

FIG. 9: Immunohistochemistry

Immunohistochemistry (IHC) staining for 3 main Damage AssociatedMolecular Proteins (DAMPs) in response to ECT treatment. Groups shown:Untreated (Untx), Electroporation (EP), Electrochemotherapy (ECT).Calreticulin (CRT), High Mobility Group Box Protein 1 (HMGB1) and HeatShock Protein 90 (HSP90).

FIG. 10: Tumour growth and survival in metastatic lung cancer model(LLC) FIG. 10a —tumour volume; FIG. 10b —Survival.

SUMMARY OF ASPECTS OF THE INVENTION

In a first aspect the present invention provides a method for thetreatment of cancer in a subject which comprises the steps of:

-   -   (i) administering electroporation to a tumour in the subject;        and    -   (ii) administering a T or B-cell activating agent to the        subject,

wherein step (i) and (ii) may be performed in either order.

The method may further comprise the administration of calcium and/or achemotherapeutic agent selected from a list comprising: alkylatingagents, nitrosoureas, ethylenimines/methylmelamine, alkyl sulfonates,antimetabolites, pyrimidine analogs, epipodophylotoxins, enzymes such asL-asparaginase; biological response modifiers such as IFNα, IL-2, G-CSFand GM-CSF; platinum coordination complexes such as cisplatin andcarboplatin, bleomycin, anthracenediones, substituted urea such ashydroxyurea, methylhydrazine derivatives including N-methylhydrazine(MIH) and procarbazine, adrenocortical suppressants such as mitotane(o,p′-DDD) and aminoglutethimide; hormones and antagonists includingadrenocorticosteroid antagonists such as prednisone and equivalents,dexamethasone and aminoglutethimide; progestin such ashydroxyprogesterone caproate, medroxyprogesterone acetate and megestrolacetate; estrogen such as diethylstilbestrol and ethinyl estradiolequivalents; antiestrogen such as tamoxifen; androgens includingtestosterone propionate and fluoxymesterone/equivalents; antiandrogenssuch as flutamide, gonadotropin-releasing hormone analogs andleuprolide; and non-steroidal antiandrogens such as flutamide.

The chemotherapeutic agent may be cisplatin or bleomycin oralternatively another agent capable of inducing necrotic or apoptoticcell death after cell electroporation e.g. CaCl₂.

The T or B cell activating agent may be selected from a list comprising:inducible Cell co-stimulator (ICOS) agonist, lactoferrin, Iscador, CD27agonist, CD40 agonist, BTLA antagonist, CD30 antagonist, ReceptorActivator of Nuclear Factor κB (RANK) agonist, CD2 agonist, OX40agonist, 4-1BB (CD137) agonist, phosphatidyserine antibodies, toll-likereceptor (TLR) agonist, interleukin (IL)-2, interferon (IFN)-α, IFN-β,IFN-γ.

The T or B cell activating agent may be an ICOS agonist.

The method may involve the administration of a combination of T or Bcell activating agents. The combination of T or B cell activating agentsmay be an ICOS agonist and IL-2.

The T or B cell activating agent may be administered systemically to thesubject. The

T or B cell activating agent may be administered by subcutaneous (SC),intraperitoneal (IP) or intravenous (IV) administration.

The T or B cell activating agent may be administered by intra-tumoural(IT) administration.

The T or B cell activating agent may be administered by pulseadministration.

The electroporation may be administered prior to administration of the Tor B cell activating agent.

The T or B cell activating agent may be administered up to 72 hoursafter the electroporation.

The interstitial pressure of the tumour may be lowered prior to orsimultaneously to administration of the T or B cell activating agent.The interstitial pressure of the tumour may be lowered by a vacuum, asonic wave or administration of an enzyme.

The T or B cell activating agent may be administered using a ‘luer lock’administration.

The cancer may be a solid tumour. The solid tumour may be selected froma list comprising: gastrointestinal cancer; malignant melanoma; head andneck malignancies; squamous cell carcinoma; breast carcinoma; prostatecancer; lung cancer; glioblastoma; bladder cancer; cervical cancer;chordoma; kidney cancer; liver cancer; ovarian cancer; pancreaticcancer; sarcoma; thyroid cancer; testicular cancer; uterine cancer;urethral cancer; or vulvar cancer.

In a second aspect, the present invention provides a T or B cellactivating agent for use in treating cancer according to the method thefirst aspect of the invention.

In a third aspect, the present invention provides a chemotherapeuticagent for use in treating cancer according to the method of the firstaspect of the invention, in which the electroporation comprisesadministration of a chemotherapeutic agent.

In a fourth aspect, the present invention relates to the use of a T or Bcell activating agent in the manufacture of a medicament for treatingcancer according to the method of the first aspect of the invention.

In a fifth aspect, the present invention relates to the use of achemotherapeutic agent in the manufacture of a medicament agent fortreating cancer according to the method of the first aspect of theinvention, in which the electroporation comprises administration of achemotherapeutic agent.

In a sixth aspect, the present invention provides a kit which comprisesa T or B cell activating agent and a chemotherapeutic agent for use intreating cancer according to the method of the first aspect, in whichthe electroporation comprises administration of a chemotherapeuticagent.

Thus, the present invention is based on a combination of electroporationand administration of a T or B-cell activating agent, which facilitatesthe activation of immune cells and the generation of an effective tumourantigen specific immune response. The combinatorial approach provided bythe present invention results in increased survival levels compared toeither chemotherapy or electroporation alone.

DETAILED DESCRIPTION

The present invention relates to a method for the treatment of cancer ina subject which comprises the steps of:

-   -   (i) administering electroporation to a tumour in the subject;        and    -   (ii) administering a T or B-cell activating agent to the        subject,

wherein step (i) and (ii) may be performed in either order.

As used herein, the term ‘treatment’/‘to treat’ refers to performing themethod on a subject in need of treatment in order to lessen, reduce orimprove at least one symptom associated with the disease and/or to slowdown, reduce or block the progression of the disease.

In the method of the present invention, the electroporation (step (i))and T or B-cell therapy (step (ii)) may be performed in either order.That is, the method may comprise step (i), followed by step (ii) or step(ii), followed by step (i).

Electroporation

As used herein, the term ‘electroporation’ refers to the delivery ofelectrical fields/pulses to a solid tumour in a subject.

The standard treatment protocol for reversible electroporation involvesthe delivery of eight 100 μsec electric pulses (1000 v/cm, 8 square wavepulses, 100 μsec each) at a frequency of between 1 and 5 KHz.

Methods for the delivery of electrical pulses to solid tumours are wellestablished in the art and numerous studies have demonstrated thesuccessful delivery of electrical pulses to a variety of solid tumourtypes.

Irreversible Electroporation (IRE) is a phenomenon in which highelectrical fields are delivered across cells in short, micro tomillisecond pulses. These pulses create irreversible defects (pores) inthe cell membrane lipid bilayer, causing cell death through loss of cellhomeostasis. Non Thermal Irreversible Electroporation (NTIRE) operatesto selectively affect only the cell membrane lipid bilayer and sparingall the other molecules in the volume of the treated tissue. NTIREablation efficacy is dependent on electric pulse parameters (number,length, frequency, magnitude and pulse shape). The electric field effectalso depends on electrode design, cell morphology and its orientation,and extra cellular matrix properties herefore, NTIRE effect shouldtherefore be evaluated separately for different tissues.

Electric pulses can be delivered by a variety of different electrodes.For example, plate electrodes with different gap between the plates maybe used for treating small and superficial tumour nodules; needleelectrodes may be used for the treatment of thicker and deeper-seatedtumour nodules; whilst a hexagonal array of electrodes may be used forlarger nodules.

Electroporation for solid tumours uses direct currents (all unipolar)with short and intense pulses (even though in vitro, time-decayed pulsescan be used). The amplitude of the pulses depends on the tissues and onthe shape and position of the electrodes, but, in vivo, the amplitude ofthe electric pulses has to be high enough to establish an electricalfield of 400 V/cm in the area of tumour (8 pulses with duration of 100microseconds). Pulses are generally delivered at a repetition frequencyof 5000 Hz, resulting in a much less discomfort for the patient and inthe shorter duration of treatment compared to earlier treatmentprocedures. For treatment of deep-seated tumours in relative vicinity ofthe heart, pulses are synchronized with absolute refractory period ofeach heartbeat to minimize the probability of interaction of pulses withthe heart function.

The electroporation used in the method of the present invention may beperformed with any electroporation system approved for clinical use.

T or B-Cell Activating Agent

As used herein, the term ‘T or B-cell activating agent’ refers to anyentity which is capable of activating either a T-cell or B-cell.

The term ‘activate’/‘activating’, as used herein, is synonymous withinduce, enhance, upregulate and stimulate. As such, the T or B-cellactivating agent may be any agent which is capable of stimulating orupregulating the activity of a T-cell or a B-cell. The terms T-cellactivation and B-cell activation are well known in the art.

T-cells are divided into subsets. Cytolytic immune cells can be T cellsor T lymphocytes which are a type of lymphocyte that play a central rolein cell-mediated immunity. They can be distinguished from otherlymphocytes, such as B cells and natural killer cells (NK cells), by thepresence of a T-cell receptor (TCR) on the cell surface. There arevarious types of T cell, as summarised below.

Helper T helper cells (TH cells) assist other white blood cells inimmunologic processes, including maturation of B cells into plasma cellsand memory B cells, and activation of cytotoxic T cells and macrophages.TH cells express CD4 on their surface. TH cells become activated whenthey are presented with peptide antigens by MHC class II molecules onthe surface of antigen presenting cells (APCs). These cells candifferentiate into one of several subtypes, including TH1, TH2, TH3,TH17, Th9, or TFH, which secrete different cytokines to facilitatedifferent types of immune responses. The activation of these TH subsetsmay be determined be the expression of specific cell surface markers andthe expression and secretion of specific cytokines, as provided in theart.

Cytolytic T cells (TC cells, or CTLs) destroy virally infected cells andtumour cells, and are also implicated in transplant rejection. CTLsexpress the CD8 at their surface. These cells recognize their targets bybinding to antigen associated with MHC class I, which is present on thesurface of all nucleated cells. The activation of cytotoxic T cells isdependent on several simultaneous interactions between moleculesexpressed on the surface of the T cell and molecules on the surface ofthe antigen-presenting cell (APC), which provides the two signal modelfor TC cell activation. TC cell activation may be determined by therelease of the cytotoxins perforin, granzymes, and granulysin. TC cellactivation may also be determined by expression of express the surfaceprotein FAS ligand (FasL)(Apo1L)(CD95L), and other markers provided inthe art; in addition to the killing of target cells in in vitrocytotoxic killing assays.

The agent may be capable of activating a T-cell and/or a B-cell. Theagent may be capable of activating a TC cell.

A non-exhaustive list of possible T or B-cell activating agents whichmay be used in the method of the present invention includes: inducibleCell co-stimulator (ICOS) agonist, lactoferrin, Iscador, a CD27 agonist,a CD40 agonist, a B- and T-lymphocyte attenuator (BTLA) antagonist, CD30antagonist, Receptor Activator of Nuclear Factor κB (RANK) agonist, CD2agonist, OX40 agonist, 4-1BB (CD137) agonist, phosphatidyserineantibodies, toll-like receptor (TLR) agonist, interleukin (IL)-2,interferon (IFN)-α, IFN-β and IFN-γ

The term ‘agonist’ is used herein to refer to an agent which is capableof inducing, stimulating or upregulating the activity of its target. Asan illustration, an ICOS agonist is an agent which is capable ofstimulating or upregulating the activity of ICOS, for example byinducing signalling through ICOS or increasing the level of signallingthrough ICOS.

Both small molecules and antibodies (in particular monoclonalantibodies) may act as agonists for a given target. Thus, in oneembodiment, an agonist as described above is a monoclonal antibody.Examples of agonist monoclonal antibodies (such as anti-ICOS monoclonalantibodies having agonist properties) will be familiar to a personskilled in the art.

The term ‘antagonist’ is used herein to refer to an agent which iscapable of blocking, preventing or downregulating the activity of itstarget. As an illustration, a BTLA antagonist is an agent is an agentwhich is capable of preventing or downregulating the activity of BTLA.For example, by preventing signalling through BTLA or reducing the levelof signalling through BTLA.

Antagonists for a given target may also be small molecules or antibodies(in particular monoclonal antibodies). Thus, in one embodiment, anantagonist as described above is a monoclonal antibody. Examples ofantagonist monoclonal antibodies will be familiar to a person skilled inthe art.

A T or B-cell activating agent which is suitable for use in the methodof the present invention may therefore by any agent which is capable ofincreasing the activation/activity of a T-cell or a B-cell compared tothe level of activation/activity in the absence of the agent.

It is well known in the art that the activation of immune cells,including T-cells and B-cells, is controlled by a balance betweenpositive activation signals (i.e. inducible Cell co-stimulator (ICOS)signalling) and negative repressive signals (i.e. B- and T-lymphocyteattenuator (BTLA) signalling). Dominance of positive activation signalsleads to immune cell activation, whilst dominance of negative repressivesignals prevents immune cell activation.

The T or B-cell activating agent for use in the method of the presentinvention may be an agent which activates a T-cell or a B-cell bystimulating/activating a positive activation signal (i.e. rather thaninhibiting/downregulating a negative repressive signal).

A non-exhaustive list of such T or B-cell activating agents includes:ICOS agonist, lactoferrin, Iscador, CD27 agonist, RANK agonist, CD2agonist, OX40 agonist, 4-1BB agonist, phosphatidyserine antibodies,toll-like receptor (TLR) agonist, interleukin (IL)-2, interferon(IFN)-α, IFN-β and IFN-γ.

In certain embodiments, the method of the present invention may comprisethe administration of a combination of T or B-cell activating agents.For example, the method may comprise the administration of at least two,at least three or a plurality of T or B-cell activating agents. Themethod may comprise the administration of at least one T-cell activatingagent and at least one B-cell activating agent. The method may comprisethe administration of at least two T-cell activating agents. The methodmay comprise the administration of at least two B-cell activatingagents.

The combination of T or B cell activating agents may be an ICOS agonistand IL-2.

Administration

The T or B-cell activating agent for use in the method of the presentinvention may be administered systemically to the subject.

For example the agent may be administered by intravenous (i.v),intraperitoneal (i.p), intra-arterial, intraventricular, intraepidural,oral or nasal administration.

The T or B-cell activating agent may be administered to the subject byintratumoural administration.

The therapeutically effective amount of one or more T or B-cellactivating agents for use in the method of the present invention can bedetermined by the ordinarily-skilled artisan with consideration ofindividual differences in age, weight, and the condition of the subject.The agents are administered to a subject (e.g. a mammal, such as ahuman) in an effective amount, which is an amount that produces adesirable result in a treated subject (e.g. the slowing or remission ofa cancer). Therapeutically effective amounts can be determinedempirically by those of skill in the art.

The T or B-cell activating agent may be administered prior to,simultaneously to, or following the administration of theelectroporation.

The T or B-cell activating agent may be administered up to 168, up to120, up to 72, up to 48, up to 24, up to 12, up to 6, up to 4 or 2 hoursprior to the administration of the electroporation.

The T or B-cell activating agent may be administered from less than twohours before to less than two hours following the administration of theelectroporation. As used herein, this time range refers to thesimultaneous administration of the T or B-cell activating agent and theelectroporation.

The T or B-cell activating agent may be administered up to 168, up to120, up to 72, up to 48, up to 24, up to 12, up to 6, up to 4 or 2 hoursfollowing the administration of the electroporation.

In certain embodiments, the T or B-cell activating agent may beadministered by pulse administration. As such, the T or B-cellactivating agent may be administered at several (i.e. more than one)time-points following the administration of the electroporation.

For example, the T or B-cell activating agent may be administered atleast once, at least twice, up to a plurality of occasions following theelectroporation. The T or B-cell activating agent may be administered at6, 12, or 24 hour intervals, or up to several days and weeks apart,targeting the optimal period for immune cell antigen presentationfacilitated by the electroporation treatment.

The T or B-cell activating agent may be administered simultaneously tothe electroporation, followed by further administrations at 24 hours, 48hours and 72 hours following electroporation. The T or B-cell activatingagent may be administered simultaneously to the electroporation,followed by further administrations at 24 hours and 72 hours followingelectroporation.

The T or B-cell activating agent may be administered 24 hours, 48 hoursand 72 hours following the electroporation. The T or B-cell activatingagent may be administered 24 hours and 72 hours following theelectroporation.

The T or B-cell activating agent may be administered using a ‘leur lock’administration. A ‘leur lock’ administration is a standardized system ofsmall-scale fluid fittings used for making leak-free connections betweena male-taper fitting and its mating female part on medical andlaboratory instruments, including hypodermic syringe tips and needles orstopcocks and needles.

There are two varieties of Luer connections: Luer-Lock and Luer-Slip.Luer-Lock fittings are securely joined by means of a tabbed hub on thefemale fitting which screws into threads in a sleeve on the malefitting. Luer-Slip fittings simply conform to Luer taper dimensions andare pressed together and held by friction (they have no threads). Luercomponents are manufactured either from metal or plastic and areavailable from many companies worldwide.

Interstitial Pressure

In certain embodiments, the interstitial pressure of the tumour islowered prior to administration of the T or B-cell activating agent.

The ‘interstitial pressure of the tumour’ refers to hydrostatic pressurein the interstitial fluid which surrounds the tumour cells. Tumours canhave high positive interstitial pressure throughout the interior, whilepressure in the outermost areas remains at close to normal physiological(marginally negative) levels. This negatively impacts on the flow byconvection of molecules from the capillaries around the tumour into thetumour interstitial spaces.

A non-exhaustive, illustrative list of tumours which have been shown tohave high interstitial pressure includes; renal cell carcinoma, cervicalcarcinoma, colorectal liver metastases, head and neck carcinoma, breastcarcinoma, metastatic carcinoma and lung carcinoma.

The interstitial pressure of a tumour may be lowered using methods knownin the art. For example, the interstitial pressure may be lowered usinga vacuum, an enzyme or sonic waves.

Vacuum—The use of a vacuum reduces the liquid stress. It creates anartificial lymphatic drainage system which reduces fluid/liquid stressand causes negative pressure to be formed.

Enzyme—The use of enzymes reduces the solid stress: it is not a fluidbased approach. The microenvironment in tumors is different than that ofnormal tissue. There is an increased number of fibroblasts in tumorstroma that stimulates the expansion of ECM and increase the matrixtension. This is due to the synthesis of abnormally large amount ofcollagen fibers, hyaluronan, GAGs, proteoglycans, and proteolyticenzymes and its inhibitors. Examples of enzymes include collagendegrading enzymes and Hyaluronan.

Sonic wave approaches, such as ultrasound—The soundwaves are transmittedas an alternation series of compressions (zones of high pressure) andrarefractions (zones of low pressure). Ultrasound reduces tumourinterstitial pressure due to mechanical (cavitation, radiation pressure)and thermal effects. Thermal effects cause damage to tumor cells and ECMwhich increase the interstitial hydraulic conductivity, reduce matrixtension and enhance tumor blood flow.

Sonic waves produced by medical ultrasound devices may also be used tolower the interstitial pressure in a tumour. Ultrasound exposures may beprovided using focused transducers which allow ultrasound waves to befocused onto very small volumes, which greatly increases theirintensity—high intensity focused ultrasound (HIFU). Focused beams arecreated using spherically-curved transducers, allowing energy to bedeposited deep inside the body.

Electrochemotherapy

The method of the present invention may further comprise administrationof calcium and/or a chemotherapeutic agent to a subject in need oftreatment.

The administration of a chemotherapeutic agent in combination withelectroporation is termed ‘electrochemotherapy’ (ECT). ECT allows thedelivery of non-permeant drugs to the cell interior. It is based on thelocal application of short and intense electric pulses that transientlypermeabilize the cell membrane, thus allowing transport of moleculesotherwise not permitted by the membrane. With the delivery of theelectric pulses, cells are subjected to an electric field that causesthe formation of nanoscale defects on the cell membrane, which alter thepermeability of the membrane. At this stage and for some time afterpulses are delivered, molecules of the cytotoxic agents can freelydiffuse into the cytoplasm and exert their cytotoxic effect. Multiplepositioning of the electrodes, and subsequent pulse delivery, can beperformed during a session to treat the whole lesion, provided that drugconcentration is sufficient. Treatment can be repeated over the courseof weeks or months to achieve regression of large lesions.

A chemotherapeutic agent contemplated includes, without limitation,alkylating agents, nitrosoureas, ethylenimines/methylmelamine, alkylsulfonates, antimetabolites, pyrimidine analogs, epipodophylotoxins,enzymes such as L-asparaginase; biological response modifiers such asIFNα, IL-2, G-CSF and GM-CSF; platinium coordination complexes such ascisplatin and carboplatin, anthracenediones, substituted urea such ashydroxyurea, methylhydrazine derivatives including N-methylhydrazine(MIH) and procarbazine, adrenocortical suppressants such as mitotane(o,p′-DDD) and aminoglutethimide; hormones and antagonists includingadrenocorticosteroid antagonists such as prednisone and equivalents,dexamethasone and aminoglutethimide; progestin such ashydroxyprogesterone caproate, medroxyprogesterone acetate and megestrolacetate; estrogen such as diethylstilbestrol and ethinyl estradiolequivalents; antiestrogen such as tamoxifen; androgens includingtestosterone propionate and fluoxymesterone/equivalents; antiandrogenssuch as flutamide, gonadotropin-releasing hormone analogs andleuprolide; and non-steroidal antiandrogens such as flutamide.

The chemotherapeutic agent may be cisplatin or bleomycin.

Alternatively CaCl₂ may be used to induce tumour necrosis postelectroporation.

Cisplatin is also known as cisplatinum, or cis-diamminedichloroplatinum(CDDP). It was the first member of a class of platinum-containinganti-cancer drugs, which now also includes carboplatin and oxaliplatin.These platinum complexes react in vivo, binding to and causingcrosslinking of DNA, which ultimately triggers apoptosis (programmedcell death).

Bleomycin is a glycopeptide antibiotic produced by the bacteriumStreptomyces verticillus. Bleomycin refers to a family of structurallyrelated compounds. The chemotherapeutical forms are primarily bleomycinA2 and B2, which function by inducing breaks in DNA.

The chemotherapeutic agent may be administered to the subject via asystemic administration method. For example, the agent may beadministered by intravenous (i.v), intraperitoneal (i.p),intra-arterial, intraventricular, intraepidural, oral or nasaladministration.

The chemotherapeutic agent may be administered to the subject byintratumoural.

The therapeutically effective amount of one or more chemotherapeuticagents for use in the method of the present invention can be determinedby the ordinarily-skilled artisan with consideration of individualdifferences in age, weight, and the condition of the mammal. The agentsare administered to a subject (e.g. a mammal, such as a human) in aneffective amount, which is an amount that produces a desirable result ina treated subject (e.g. the slowing or remission of a cancer).Therapeutically effective amounts can be determined empirically by thoseof skill in the art.

The timing of the administration of the chemotherapeutic agent withrespect to the electroporation may be performed according to the methodsknown in the art.

For example, the chemotherapeutic agent may be administered prior to,simultaneously to, or post the administration of the electroporation.

The chemotherapeutic agent may be administered up to 168, up to 120, upto 72, up to 48, up to 24, up to 12, up to 6, up to 4 or 2 hours priorto the administration of the electroporation.

The chemotherapeutic agent may be administered from less than two hoursbefore to less than two hours following the administration of theelectroporation. As used herein, this time range refers to thesimultaneous administration of the chemotherapeutic agent and theelectroporation.

The chemotherapeutic agent may be administered up to 168, up to 120, upto 72, up to 48, up to 24, up to 12, up to 6, up to 4 or 2 hours postthe administration of the electroporation.

Cancer

The method of the present invention is used to treat cancer.

The method may be used to treat a solid tumour. The term ‘solid tumour’is used herein to refer to a malignant neoplasm. Thus it includes allcancers, other than leukaemia.

A non-exhaustive, illustrative list of solid tumours which may betreated by the method of the present invention includes;gastrointestinal cancer; malignant melanoma; head and neck malignancies;squamous cell carcinoma; breast carcinoma; prostate cancer; lung cancer;glioblastoma; bladder cancer; cervical cancer; chordoma; kidney cancer;

liver cancer; ovarian cancer; pancreatic cancer; sarcoma; thyroidcancer; testicular cancer; uterine cancer; urethral cancer; and vulvarcancer.

Use

The present invention also provides a T or B-cell activating agent, asdefined herein, for use in treating cancer according to the method ofthe first aspect of the invention.

The present invention further relates to the use of a T or B-cellactivating agent, as defined herein, in the manufacture of a medicamentfor treating cancer according to the method of the first aspect of theinvention.

Kit

The present invention also provides a kit which comprises a T or B-cellactivating agent and a chemotherapeutic agent, as defined herein, foruse in treating cancer according to the method of the first aspect ofthe invention, in which the electroporation comprises administration ofthe chemotherapeutic agent.

The invention will now be further described by way of Examples, whichare meant to serve to assist one of ordinary skill in the art incarrying out the invention and are not intended in any way to limit thescope of the invention.

EXAMPLES Example 1 Effect of ECT Combined with ICOS on Tumour Growth inCT26 Model

Balb/C mice were subcutaneously injected with 1×10⁶ CT26 cells. Whentumours reached an approximate size of 100 mm³ they were treated withelectroporation only, electrochemotherapy only (ECT),electrochemotherapy combined with ICOS or untreated (FIG. 1).

Example 2 Effect of ECT Combined with ICOS on Survival in CT26 Model

Representative Kaplan-Meier survival curve of CT26 treated tumours wasmeasured. Only mice treated with ICOS/ECT combination survived as 40% ofmice survived and were still alive at approx. 200 days. All other groupswere sacrificed by day 38 (FIG. 2)

Example 3 Effect of ECT Combined with ICOS on Tumour Growth in B16F10Model

C57BL/6J mice were subcutaneously injected with 2×10⁵ B16F10 cells inthe flank. When the tumours grew to an approximate size of 100 mm³ theywere treated with electroporation only, electrochemotherapy only,electrochemotherapy combined with ICOS, ICOS only or untreated (FIG. 3).

Example 4 Effect of ECT Combined with ICOS on Survival in B16F10 Model

Representative Kaplan-Meier survival curve of B16F10 treated tumours wasmeasured. Mice treated with ICOS/ECT combination survived to 48 days.All other groups were sacrificed by day 25 (FIG. 4).

Example 5 Effect of ECT Combined with ICOS on Lung Tumour Size

The effect of ECT combined with ICOS was examined in a metastatic lungcancer model. Lung weight is a physical indicator for the presence oflung metastases. Both the untreated (Untx) and electroporation (EP)groups had lung weights almost four times that of the healthy control,indicating a high metastases burden in the lungs. Lung weights in theElectrochemotherapy (ECT) and iCOS groups showed a slight increasecompared to the healthy control, but the combinational group (iCOS plusECT) exhibited a lung weight comparable to that of the healthy control.The significance of this is that only the primary tumours of the animalswere treated but the treatment was also successful againstsecondary/distal tumours (FIG. 5).

Example 6 Cell Histology

Programmed Cell Death Receptor 1 (PD-1) and Programmed Cell Death Ligand(PD-L1) are molecules belonging to the B7 superfamily, which mediatesimmune cell responses towards cancer cells. When PD-1 and PD-L1 bindwithin the tumour microenvironment, it causes T cell energy byinterfering with key immune signalling and so both PD-1 and PD-L1expression within tumour tissue are negative prognostic markers. Highlevels of both staining were shown in the Untx and ECT groups and eventhe PD-1 receptor staining was quite high for the iCOS only group.However there was a dramatic decrease in expression of both aftertreatment with iCOS plus ECT. If PD-1 and PD-L1 are not expressed thenthey can no longer cause T cell energy and a more prolonged immuneresponse can be achieved (FIG. 6).

Example 7 Flow Cytometry Analysis

Flow cytometry analysis was carried out on excised tumour tissue. Thisanalysis showed significant increases in the following immune cellpopulations in response to the ECT and immunotherapy combinations: CD8+T cells (key immune cells that target and kill tumour cells), B220+ Bcells (responsible for immune cell signalling), and CD138+ memory Bcells (B cells that produce antibodies specific to the tumour, allowingfor the generation of immune memory against the tumour) (FIGS. 7-8).

Example 8 Immunohistochemistry

Immunohistochemistry (IHC) staining was performed for three main DamageAssociated Molecular Proteins (DAMPs), in response to ECT treatment.DAMPs play key roles in immune activation when expressed extracellularlyby interacting with Antigen Presenting Cells (APCs). ECT caused dramaticincreases in the levels of Calreticulin (CRT), High Mobility Group BoxProtein 1 (HMGB1) and Heat Shock Protein 90 (HSP90). This indicates thatECT alone is sufficient to prime the immune system and generate a weakbut positive immune response, that can then be potentiated withcombination immunotherapy (FIG. 9).

Example 9 Metastatic Lung Cancer Model

Tumour growth and animal survival were analysed in the metastatic lungcancer model. Treatment with combination immunotherapy (iCOS plus ECT)produced significant inhibition in tumour growth and significantincreases in animal survival (40-60% cures across multiple experimentsand tumour models) (FIG. 10).

Materials and Methods Cell Tissue Culture

Tumour cell lines CT26 and B16F10 were obtained from the American TypeCell Collection (Manassas, Va.). The murine colon adenocarcinoma, CT26was cultured with Dulbecco's modified Eagle's media (Sigma) supplementedwith 10% v/v fetal calf serum, 300 μg/ml L-glutamine. The mouse melanomaB16F10 cell line was cultured in RPMI-1640 (Sigma) supplemented with 10%v/v fetal calf serum and 300 μg/ml L-glutamine. Cells were maintained inlogarithmic phase growth at 37° C. in a humidified atmospheresupplemented with 5% CO2.

Animals and Tumour Induction

Female Balb/c and C57BL/6J (6-8 weeks) were obtained from HarlanLaboratories (Oxfordshire, England). For routine tumour induction, 1×10⁶CT26 and 2×10⁵ B16F10 tumour cells suspended in 200 μl of serum freeDMEM were injected subcutaneously into the flank of the female Balb/C orC57 BL/6J. Following tumour establishment, tumours were allowed to growand develop and were monitored by alternate day measurements in twodimensions using vernier callipers. Tumour volume was calculatedaccording to the formula V=ab²π/6, where a is the longest diameter ofthe tumour and b is the longest diameter perpendicular to diameter a.From these volumes, tumour growth curves were constructed. Miceeuthanized when the tumour diameter was between 1.7 cm³.

Ethics Statement

All murine husbandry and experimental procedures were approved by theUniversity College Cork Animal Experimentation Ethics Committee andcarried out under licenses issued by the Department of Health, Irelandas directed by the Cruelty to Animals Act Ireland and EU StatutoryInstructions.

In Vivo Electroporation

Once tumours reached a size of 0.3 cm×0.3 cm, electrochemotherapy wasadministered using an established, standard operation procedure.Procedures were performed under local anaesthesia, a combination ofketamine (2-10 mg/kg) and Xylazine (0.05-0.1 mg/kg) in 100 uL of sterilePBS, was administered via intra-peritoneal injection. The intra-tumouraldose of Cisplatin was set at 5 mg/kg in 200 ul sterile PBS and wasadministered pre electroporation. Electric pulses were generated by theCliniporator (IGEA, Carpi, Italy) (8 square wave pulses 1000 V/cm for100 μs at 5 kHz) and were delivered into the tumours using 3 pairs oflinear needle electrodes.

Chemotherapeutic Drugs

Cisplatin: is a chemotherapy drug. It was the first member of a class ofplatinum-containing anti-cancer drugs, which now also includescarboplatin and oxaliplatin. These platinum complexes react in vivo,binding to and causing crosslinking of DNA, which ultimately triggersapoptosis.

Antibody

ICOS antibody: A member of the costimulatory molecule family, induciblecostimulator (ICOS), is expressed on activated T or B cells and plays acritical role in their primary activation and cytokine production. Thefollowing anti-ICOS monoclonal antibodies were employed: Clone C398.4A,Biolegend; Clone 27A12, BioXCell.

Treatment Schedule

Day 0 ECT, Day 3: ICOS administration, Day 6: ICOS administration Day 9:ICOS administration.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inimmunology, cardiac biology or related fields are intended to be withinthe scope of the following claims.

1. A method for the treatment of cancer in a subject which comprises thesteps of: (i) administering electroporation to a tumour in the subject;and (ii) administering a T or B cell activating agent the subject,wherein step (i) and (ii) may be performed in either order.
 2. A methodaccording to claim 1 wherein step (i) further comprises administrationof calcium.
 3. A method according to claim 1 wherein step (i) furthercomprises administration of a chemotherapeutic agent selected from alist comprising: alkylating agents, nitrosoureas,ethylenimines/methylmelamine, alkyl sulfonates, antimetabolites,pyrimidine analogs, epipodophylotoxins, enzymes such as L-asparaginase;biological response modifiers such as IFNα, IL-2, G-CSF and GM-CSF;platinium coordination complexes such as cisplatin and carboplatin,bleomycin, anthracenediones, substituted urea such as hydroxyurea,methylhydrazine derivatives including N-methylhydrazine (MIH) andprocarbazine, adrenocortical suppressants such as mitotane (o,p′ -DDD)and aminoglutethimide; hormones and antagonists includingadrenocorticosteriod antagonists such as prednisone and equivalents,dexamethasone and aminoglutethimide; progestin such ashydroxyprogesterone caproate, medroxyprogesterone acetate and megestrolacetate; estrogen such as diethylstillbestrol and ethinyl estradiolequivalents; antiestrogen such as tamoxifen; androgens includingtestosterone propionate and fluoxymesterone/equivalents; antiandrogenssuch as flutamide, gonadotropin-releasing hormone analogs andleuprolide; and non-steroidal antiandrogens such as flutamide.
 4. Amethod according to claim 2 wherein the chemotherapeutic agent iscisplatin or bleomycin.
 5. A method according to claim 1 wherein the Tor B cell activating agent is selected from a list comprising: inducibleCell co-stimulator (ICOS) agonist, lactoferin, Iscador, CD27 agonist,CD40 agonist, BTLA antagonist, CD30 antagonist, Receptor Activator ofNuclear Factor κB (RANK) agonist, CD2 agonist, OX40 agonist, 4-1 BB(CD137) agonist, phosphatidyserine antibodies, toll-like receptor (TLR)agonist, interleukin (IL)-2, interferon (IFN)-α IFN-β, IFN-γ.
 6. Amethod according to claim 5 wherein the T or B cell activating agent isan ICOS agonist.
 7. A method according to claim 1 in which a combinationof T or B cell activating agents are administered.
 8. A method accordingto claim 7 wherein the combination of agents comprise an ICOS agonistand IL-2.
 9. A method according to claim 1 in which the T or B cellactivating agent is administered systemically to the subject.
 10. Amethod according to claim 9 in which the T or B cell activating agent isadministered by subcutaneous (SC), intraperitoneal (IP) or intravenous(IV) administration.
 11. A method according to claim 1 in which the T orB cell activating agent is administered by intra-tumoural (IT)administration.
 12. A method according to claim 1 wherein theelectroporation and T or B cell activating agent are administered bypulse administration.
 13. A method according to claim 1 wherein theelectroporation is administered prior to administration of the T cellactivating agent.
 14. A method according to claim 13 in which the T or Bcell activating agent is administered up to 72 hours after theelectroporation.
 15. A method according to claim 1 wherein theinterstitial pressure of the tumour is lowered prior or simultaneouslyto administration of the T or B cell activating agent.
 16. A methodaccording to claim 15 wherein the interstitial pressure of the tumour islowered by a vacuum, a sonic wave or administration of an enzyme.
 17. Amethod according to claim 1 wherein the T or B cell activating agent isadministered using a ‘luer lock’ administration.
 18. A method accordingto claim 1 wherein the cancer is a solid tumour.
 19. A method accordingto claim 18 wherein the solid tumour is selected from a list comprising:gastrointestinal cancer; malignant melanoma; head and neck malignancies;squamous cell carcinoma; breast carcinoma; prostate cancer; lung cancer;glioblastoma; bladder cancer; cervical cancer; chordoma; kidney cancer;liver cancer; ovarian cancer; pancreatic cancer; sarcoma; thyroidcancer; testicular cancer; uterine cancer; urethral cancer; or vulvarcancer.
 20. A kit comprising a T or B cell activating agent and calciumfor use in treating cancer.